Limits...
Importin α/β mediates nuclear import of individual SUMO E1 subunits and of the holo-enzyme.

Moutty MC, Sakin V, Melchior F - Mol. Biol. Cell (2011)

Bottom Line: Here we show that the mammalian E1 subunits can be imported separately, identify nuclear localization signals (NLSs) in Aos1 and in Uba2, and demonstrate that their import is mediated by importin α/β in vitro and in intact cells.Once assembled into a stable heterodimer, the E1 enzyme can still be efficiently imported by importin α/β, due to the Uba2 NLS that is still accessible.These pathways may serve distinct purposes: import of nascent subunits prior to assembly and reimport of stable E1 enzyme complex after mitosis.

View Article: PubMed Central - PubMed

Affiliation: Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Alliance, Heidelberg, Germany. f.melchior@zmbh.uni-heidelberg.de

ABSTRACT
SUMOylation, reversible attachment of small ubiquitin-related modifier (SUMO), serves to regulate hundreds of proteins. Consistent with predominantly nuclear targets, enzymes required for attachment and removal of SUMO are highly enriched in this compartment. This is true also for the first enzyme of the SUMOylation cascade, the SUMO E1 enzyme heterodimer, Aos1/Uba2 (SAE1/SAE2). This essential enzyme serves to activate SUMO and to transfer it to the E2-conjugating enzyme Ubc9. Although the last 40 amino acids in yeast Uba2 have been implicated in its nuclear localization, little was known about the import pathways of Aos1, Uba2, and/or of the assembled E1 heterodimer. Here we show that the mammalian E1 subunits can be imported separately, identify nuclear localization signals (NLSs) in Aos1 and in Uba2, and demonstrate that their import is mediated by importin α/β in vitro and in intact cells. Once assembled into a stable heterodimer, the E1 enzyme can still be efficiently imported by importin α/β, due to the Uba2 NLS that is still accessible. These pathways may serve distinct purposes: import of nascent subunits prior to assembly and reimport of stable E1 enzyme complex after mitosis.

Show MeSH
Two mechanisms for generation of active nuclear E1 complex. (A) The single subunits Aos1 and Uba2 are independently imported by importin α/β via their distinct NLSs. After translocation into the nucleus, active SUMO E1 complex is formed by heterodimerization. (B) Alternatively, E1 enzyme assembles in the cytoplasm, or reaches the cytoplasm upon nuclear envelope breakdown, and is imported as a complex by importin α/β. This mechanism requires the NLS of Uba2, whereas the NLS of Aos1 is masked.
© Copyright Policy - creative-commons
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3046061&req=5

Figure 8: Two mechanisms for generation of active nuclear E1 complex. (A) The single subunits Aos1 and Uba2 are independently imported by importin α/β via their distinct NLSs. After translocation into the nucleus, active SUMO E1 complex is formed by heterodimerization. (B) Alternatively, E1 enzyme assembles in the cytoplasm, or reaches the cytoplasm upon nuclear envelope breakdown, and is imported as a complex by importin α/β. This mechanism requires the NLS of Uba2, whereas the NLS of Aos1 is masked.

Mentions: The findings described here reveal two pathways for the generation of intranuclear E1 enzyme, both of which depend on importin α/β (Figure 8). First, both subunits contain an NLS and can be transported separately, prior to heterodimerization in the nucleus (left). Second, E1 complexes may already assemble in the cytoplasm, prior to being imported. Although the NLS in Aos1 is not functional in the heterodimer, the NLS in Uba2 is available for recognition and import by importin α/β (right). Because E1 complexes don’t seem to dissociate in cells, the latter pathway is required at least for reimport of the E1 complex after mitosis.


Importin α/β mediates nuclear import of individual SUMO E1 subunits and of the holo-enzyme.

Moutty MC, Sakin V, Melchior F - Mol. Biol. Cell (2011)

Two mechanisms for generation of active nuclear E1 complex. (A) The single subunits Aos1 and Uba2 are independently imported by importin α/β via their distinct NLSs. After translocation into the nucleus, active SUMO E1 complex is formed by heterodimerization. (B) Alternatively, E1 enzyme assembles in the cytoplasm, or reaches the cytoplasm upon nuclear envelope breakdown, and is imported as a complex by importin α/β. This mechanism requires the NLS of Uba2, whereas the NLS of Aos1 is masked.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3046061&req=5

Figure 8: Two mechanisms for generation of active nuclear E1 complex. (A) The single subunits Aos1 and Uba2 are independently imported by importin α/β via their distinct NLSs. After translocation into the nucleus, active SUMO E1 complex is formed by heterodimerization. (B) Alternatively, E1 enzyme assembles in the cytoplasm, or reaches the cytoplasm upon nuclear envelope breakdown, and is imported as a complex by importin α/β. This mechanism requires the NLS of Uba2, whereas the NLS of Aos1 is masked.
Mentions: The findings described here reveal two pathways for the generation of intranuclear E1 enzyme, both of which depend on importin α/β (Figure 8). First, both subunits contain an NLS and can be transported separately, prior to heterodimerization in the nucleus (left). Second, E1 complexes may already assemble in the cytoplasm, prior to being imported. Although the NLS in Aos1 is not functional in the heterodimer, the NLS in Uba2 is available for recognition and import by importin α/β (right). Because E1 complexes don’t seem to dissociate in cells, the latter pathway is required at least for reimport of the E1 complex after mitosis.

Bottom Line: Here we show that the mammalian E1 subunits can be imported separately, identify nuclear localization signals (NLSs) in Aos1 and in Uba2, and demonstrate that their import is mediated by importin α/β in vitro and in intact cells.Once assembled into a stable heterodimer, the E1 enzyme can still be efficiently imported by importin α/β, due to the Uba2 NLS that is still accessible.These pathways may serve distinct purposes: import of nascent subunits prior to assembly and reimport of stable E1 enzyme complex after mitosis.

View Article: PubMed Central - PubMed

Affiliation: Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-ZMBH Alliance, Heidelberg, Germany. f.melchior@zmbh.uni-heidelberg.de

ABSTRACT
SUMOylation, reversible attachment of small ubiquitin-related modifier (SUMO), serves to regulate hundreds of proteins. Consistent with predominantly nuclear targets, enzymes required for attachment and removal of SUMO are highly enriched in this compartment. This is true also for the first enzyme of the SUMOylation cascade, the SUMO E1 enzyme heterodimer, Aos1/Uba2 (SAE1/SAE2). This essential enzyme serves to activate SUMO and to transfer it to the E2-conjugating enzyme Ubc9. Although the last 40 amino acids in yeast Uba2 have been implicated in its nuclear localization, little was known about the import pathways of Aos1, Uba2, and/or of the assembled E1 heterodimer. Here we show that the mammalian E1 subunits can be imported separately, identify nuclear localization signals (NLSs) in Aos1 and in Uba2, and demonstrate that their import is mediated by importin α/β in vitro and in intact cells. Once assembled into a stable heterodimer, the E1 enzyme can still be efficiently imported by importin α/β, due to the Uba2 NLS that is still accessible. These pathways may serve distinct purposes: import of nascent subunits prior to assembly and reimport of stable E1 enzyme complex after mitosis.

Show MeSH